D-isoglutamyl-D-tryptophan (also known as H-D-iGlu-Trp-OH or Thymodepressin) is a synthetic hemoregulatory dipeptide developed for the treatment of autoimmune diseases including psoriasis (Sapuntsova, S. G., et al., (May 2002), Bulletin of Experimental Biology and Medicine, 133(5), 488-490). Thymodepressin is considered an effective treatment for psoriasis in Russia (U.S. Pat. No. 5,736,519), where the drug is currently sold as the disodium salt in liquid formulation for injection and intranasal administration. It is an immunosuppressant and selectively inhibits proliferation of bone marrow cells and thus induces immune depression.
The known solid form of D-isoglutamyl-D-tryptophan disodium salt is an amorphous powder which is hygroscopic and very difficult to handle. The structure of thymodepressin disodium salt is described in Kashirin, D. M., et al. (2000), Pharmaceutical Chemistry Journal, 34(11), 619-622. The mono sodium salt of D-isoglutamyl-D-tryptophan is identified by the Chemical Abstracts Service (CAS) Registry System and is listed in the CAS REGISTRYSM File, but there is no publication concerning its preparation and physical properties. A powdery or amorphous form of a compound such as D-isoglutamyl-D-tryptophan, intended for pharmaceutical use may give rise to manufacturing problems due to bulk density issues, hygroscopicity and variable water content that cannot be corrected by vacuum drying. D-isoglutamyl-D-tryptophan is a dipeptide and the drying of an amorphous form at elevated temperature, for example, 80-100° C. under vacuum is not recommended.
The only synthesis of H-D-iGlu-D-Trp-OH reported in the literature is disclosed in U.S. Pat. No. 5,736,519. According to the process reported in Example 1 of U.S. Pat. No. 5,736,519, which is depicted herein as Scheme 1, Boc-D-Glu-OH (1.1) is reacted with 1,3-dicyclohexylcabodiimide (DCC) to give the cyclic anhydride (1.2). Upon removal of the dicyclohexyl urea (DCU) by filtration, the anhydride (1.2) is reacted with H-D-Trp-OH to give a mixture of the dipeptide Boc-D-iGlu-D-Trp-OH (1.3) and Boc-D-Glu-D-Trp-OH (1.4). The yield of the combined crude Boc-D-iGlu-D-Trp-OH (1.3) and Boc-D-Glu-D-Trp-OH (1.4) is 70%. However, the mixture only contains no more than 35% of the desired intermediate Boc-D-iGlu-D-Trp-OH (1.3). The Boc protective group is removed by stirring a solution of the (1.3) and (1.4) in formic acid as the solvent at 40° C. for 1 hr. The ratio of (1.3) and (1.4) to formic acid is about 1 g: 8 mL (weight to volume). The product is a mixture of H-D-iGlu-D-Trp-OH (1.5) and H-D-Glu-D-Trp-OH (1.6). Since the peptides (1.5) and (1.6) are present in equal amount, the purification requires ion exchange chromatography using pyridine acetate buffer. The yield of the desired product H-D-iGlu-D-Trp-OH (1.5) is 35% from Boc-D-iGlu-D-Trp-OH (1.3). Thus, the overall yield of H-D-iGlu-D-Trp-OH (1.5) from Boc-D-Glu-OH is 12.25%.

The process described in U.S. Pat. No. 5,736,519 has several disadvantages as follows:    1. DCC in step 1A may lead to other by-products such as
     The by-products from DCC coupling of peptides have been reported in Marder, O., and Albericio, F. (June 2003), Chemical Oggi (Chemistry Today), 6-32.    2. The deprotection of Boc-D-iGlu-D-Trp-OH (1.3) requires elevated temperature and the final purification of H-D-iGlu-D-Trp-OH requires a very toxic solvent pyridine. Elevated temperature in the deprotection of (1.3) may result in the N-tert-butyl indole derivative (1.7) as an impurity (Löw, M., et. al. (1978), Hoppe-Seyler's Z. Physiol. Chem., 359(12):1643-51). In addition, the peptide may cyclize to give the glutarimide (1.8) (Pandit, U. K. (1989), Pure & Appl. Chem., Vol. 61, No. 3, pp. 423-426).
    3. The coupling reaction only produces a 1:1 mixture of Boc-D-iGlu-D-Trp-OH (1.3) and Boc-D-Glu-D-Trp-OH (1.4). The maximum yield of (1.3) cannot exceed 50% in the coupling step 1B. A mixture of D-Glu-D-Trp-OH and D-iGlu-D-Trp-OH is formed at the end of the synthesis. The peptides must be separated by ion exchange chromatrography and reverse phase preparative high pressure liquid chromatography. The overall yield of H-D-iGlu-D-Trp-OH (1.5) is 12.25% and preparative HPLC purification is very time consuming and inefficient. The retention time for two similar isomers H-D-iGlu-D-Trp-OH (1.5) and H-D-Glu-D-Trp-OH (1.6) are not reported. Repeated cycles of separation to enrich the purity of the desired isomer (1.5) are very inefficient. This process cannot be amenable to the large scale manufacturing.    4. The opposite diastereomer L-isoglutamyl-L-tryptophan (also known as H-L-iGlu-L-Trp-OH or Bestim) is an immunostimulant (see U.S. Pat. No. 5,774,452). Bestim has been used in ulcer treatment. It decreases the inflammatory effect in the stomach and duodenal mucosa and precipitates regress of clinical symptoms and scarring of the ulcer (Tkacheva, A., et al. (2004), Eksp Klin Gastroenterol. (6):29-33, 163). The synthesis of the H-L-iGlu-L-Trp-OH, mono sodium salt (1:1) is depicted in Scheme 2 (U.S. Pat. No. 5,744,452).

In the U.S. Pat. No. 5,744,452 process, step 1 produces dicyclohexylurea as a by-product and must be removed in filtration. In the second step, the trifluoroacetic acid is claimed to have removed the γ-O-benzyl ester of the glutamic acid unit of (2.2). The benzyl ester of (2.3) is removed by transfer hydrogenation with ammonium formate, palladium catalyst, sodium bicarbonate in isopropanol at elevated temperature to give H-L-iGlu-L-Trp-OH mono sodium salt (2.4). A solid phase synthesis of (2.4) is also reported in the same patent, but the tryptophan moiety must be protected as the formamide, and later deprotected. The other diastereomers L-isoglutamyl-D-tryptophan and D-isoglutamyl-L-tryptophan are also known compounds (U.S. Pat. No. 5,916,878).
The syntheses of the H-D-iGlu-L-Trp-OH and H-L-iGlu-D-Trp-OH are reported in Scheme 3 and Scheme 4, respectively (U.S. Pat. No. 5,916,878).


The processes reported in Schemes 2, 3 and 4 may have overcome the regiospecific synthesis of gamma amide product (2.2), (3.2) and (4.2) without the formation of the alpha amide product, but they involve a hydrogenation step in the removal of a benzyl ester in compounds (2.3), (3.2) and (4.2). This requires the use of a large amount of palladium catalyst. The second concern is the partial reduction of the indole ring at the manufacturing scale. The third concern is the formation of glutarimide, 2-(3-amino-2,6-dioxo-piperidin-1-yl)-3-(1H-indol-3-yl)-propionic acid in the hydrogenation process. The fourth concern is cost. The cost of a CBz-Glu-OBzl derivative such as (3.1) and (4.1) is almost twice the price of the corresponding Boc-Glu-OBzl in fine chemicals manufacturing. The processes in schemes 3 and 4 require HPLC purification of the final product. The overall yields are 33% and 35.9%, respectively. Scheme 2 requires the use of trifluoroacetic acid, which introduces other impurities into the reaction. Furthermore, the process in Scheme 2 uses dicyclohexylcarbodiimide as a peptide coupling agent. The removal of trace amount of impurities from this reagent is a serious issue in chemical manufacturing. The technology is therefore not amenable to industrial production, and the same cannot be adopted for the large scale production of H-D-isoglutamyl-D-tryptophan.